The invention is in the field of immunoassays. More specifically, it relates to one-step lateral flow assays wherein an analyte is applied to a test strip in a sample zone and detected in a capture zone. The invention provides means for improving the speed and accuracy of such assays.
One-step nonbibulous lateral flow assays are described in WO92/12428 published Jul. 23, 1992. In the exemplified version of these assays, a sample containing analyte is applied to a sample zone in a nonbibulous matrix which permits nonchromatographic flow of solution components. The analyte travels through the labeling zone containing antibody to analyte coupled with visible label. The analyte picks up labeled antibody and the sample flows, thence, to a capture zone where the complex of analyte and labeled anti-analyte is captured and detected in a sandwich using an additional anti-analyte component adsorbed in the capture zone on a xe2x80x9ctest barxe2x80x9d. A control bar in the capture zone contains biotinylated rabbit xcex3-globulin which captures label coupled to avidin. This control label travels along with the sample into the capture zone. The background section of WO92/12428 describes and cites additional patent publications relating to other format"" for lateral flow assays.
In addition, PCT Application WO94/01775 published Jan. 20, 1994 describes similar one-step lateral flow assay procedures and devices wherein the anti-analyte in the labeling zone is coupled to a visible label in a particular way. Antibody to analyte is coupled to an enzyme that converts a substrate to a colored-dye, which dye then complexes with the enzyme/anti-analyte to provide a visible label. Again, the analyte is detected in a capture zone as a sandwich using a different antibody specifically reactive with analyte on the test bar.
The efficiency and accuracy of the self-contained, lateral flow, double-antibody sandwich immunoassays described in these PCT Applications depends not only on the ability of the label to be released readily from the labeling zone with sufficient rapidity upon contact with the sample, but also on the speed of binding of labeled anti-analyte to any analyte contained in the sample and of binding of analyte to the anti-analyte contained in the capture zone.
The general principle of such assays has also been applied to antibody detection where at least one anti-analyte may be an antigen. In this case, the sandwich contains a specific antiglobulin for the antibody to be detected, and a labeled form of the antigen for this antibody. Competitive formats for these assays have also been devised. In all of these formats, the rate of interaction of the components is significant.
The interaction of labeled anti-analyte and analyte must take place within 30 seconds or less and capture must occur rapidly as well. In the event that the labeled anti-analyte interaction with analyte and the capture of the resulting complex in the capture zone do not occur within the required times, which is often the case, a more efficient system for labeling and capture needs to be used. The present invention provides such efficient labeling and capture systems by supplementing the anti-analyte/analyte interactions with other, more efficient, specific binding pair interactions, such as the interaction of biotin and avidin or streptavidin.
Biotin/streptavidin interactions have been utilized in other ways in immunoassay procedures for some time. For example, in U.S. Pat. No. 5,126,241, streptavidin adsorbed to solid support is used to bind biotinylated antigen in a procedure which involves incubation to form a complex in which the analyte to be determined competes with label and solid support for access to an antibody capable of binding all three. U.S. Pat. No. 4,496,654 describes an assay for human chorionic gonadotropin conducted by binding biotinylated antibody to an avidin-coupled paper disk, reacting the antibody on a disk with a solution suspected of containing hCG, and then determining the amount of hCG on the disk using standard determination techniques. This assay results in a sandwich of hCG formed from anti-hCG bound to solid support through biotin/avidin linkage and labeled anti-hCG. This assay does not involve a lateral flow of sample.
U.S. Pat. No. 5,001,049 describes a method for determining antibodies against human HIV which involves incubating streptavidin-derivatized solid support with a biotinylated peptide reactive with anti-HIV, and then detecting any bound antibody with labeled antibody receptor. Again, lateral flow does not take place in these assays. RE 34,132, which is a reissue of U.S. Pat. No. 4,945,042, describes a direct assay for an antibody wherein the antibody to be determined serves as a link between a labeled epitope and an epitope bound to substrate through a streptavidin/biotin link. Again, speed of reaction is not critical, since a lateral flow format is not required.
The invention provides means to improve the efficiency of one-step lateral flow assays by expediting labeling and/or capture reactions that result in the detection of analyte, and by a novel strategy for the design of competition protocols. In general, in some of these assays, the analyte is detected as a labeled sandwich containing the analyte captured in a detection zone. In the improved methods of the invention as applied to these assays, a member of a specific binding pair with a specificity irrelevant to analyte/anti-analyte interaction is coupled the label, to a mobile anti-analyte, to the xe2x80x9ccapturexe2x80x9d anti-analyte in the nondetecting portion of the capture zone, or to a competitor to the analyte in the sample-receiving zone and is used to effect binding of label to analyte-containing complex or of complex to solid support.
Thus, in one aspect, the invention is directed to a method to determine the presence, absence or amount of an analyte in a sample in a one-step lateral flow assay conducted on a test strip. The test strip has a sample-receiving zone, an optional labeling zone, and a capture zone. The method comprises applying the sample to the sample-receiving zone, allowing the sample to proceed through the labeling zone, if present, to the capture zone and assessing the capture zone for the presence, absence or amount of label in the capture zone. The analyte complexed or coupled to the label, or a labeled competitor of the analyte, is typically captured in a detection bar in the capture zone.
In a direct assay, the analyte is captured as a sandwich comprising two anti-analytes, one of said anti-analytes is bound to the detection bar and the other anti-analyte is bound or coupled to a label. At least one of said label and the solid matrix in said detection bar is bound to an anti-analyte through members of a specific binding pair irrelevant to the specificity of the analyte/anti-analyte interaction.
In one competitive format, labeled competitor is added to the sample before the sample is applied to the sample-receiving zone, or is itself contained in the sample-receiving zone where labeled competitor competes for anti-analyte coupled to a member of the irrelevant specific binding pair, and the resulting complexes are carried to the detection zone where labeled competitor is detected; the amount of the labeled competitor in the detection zone is inversely proportional to the amount of analyte in the sample. In this format, of course, no labeling zone may be necessary.
In additional improvements included within the scope of the invention, unlabeled competitor is used in the assay. In one approach, the competitor for analyte is coupled to a member of a specific binding pair irrelevant to the analyte/anti-analyte interaction, which member permits the competitor to be captured in the detection zone by its counterpart. The competitor bearing the specific binding pair member is typically supplied in the sample-receiving zone or is mixed with the sample prior to application to the sample-receiving zone. The coupled competitor and the analyte then compete for labeled anti-analyte in the labeling zone; only the competitor (bearing the labeled anti-analyte) is captured in the detection zone; analyte bound to label is lost. Thus, again, the amount of label detected is inversely proportional to the concentration of analyte.
In still another embodiment of the competitive format, unlabeled analyte or analogous competitor behaves as a gatekeeper to prevent analyte carrying label into the detection zone. The competitor is contained in a portion of the capture zone proximal to the sample receiving zone. Binding in the detection zone may be either through anti-analyte adsorbed thereto, or through a member of a specific binding pair with specificity irrelevant to the analyte/anti-analyte interaction as described above.
In additional aspects, the invention is related to test strips for the performance of the method of the invention. Such test strips contain a sample-receiving zone, an optional labeling zone, and a capture zone, wherein at least one of said zones contains a substance that includes one member of a nonanalyte-related specific binding pair, and another zone contains the counterpart member of said nonanalyte related specific binding pair.
In embodiments wherein a competitor for anti-analyte behaves as a gatekeeper to the detecting portion of a capture zone, the invention is related to test strips having a sample-receiving zone, a labeling zone and a capture zone wherein the capture zone is divided into at least two portions; a gatekeeper portion and a detecting portion wherein said gatekeeping portion is located on the test strip between the labeling zone and the detecting portion, i.e., proximal to the sample-receiving zone. The labeling zone of said test strips contains a label coupled to anti-analyte, which anti-analyte is optionally coupled to one member of a specific binding pair with specificity irrelevant to analyte/anti-analyte interaction. The detecting zone contains a second anti-analyte or a counterpart binding pair member as appropriate.
In still another aspect, the invention relates to complexes which comprise analyte sandwiched between two anti-analyte components, at least one of said anti-analyte components being coupled to label or to a support matrix through counterpart members of a specific binding pair unrelated to analyte.